The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both these precursor (TRS and Eurasian) swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in the ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin NA and M gene segments contributed to efficient respiratory droplet transmission of the pH1N1 virus likely by modulating the release of influenza particles. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrated that the release of influenza particles into the air correlated with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus was dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus. Compared to seasonal influenza viruses, the 2009 pandemic H1N1 (pH1N1) virus caused greater morbidity and mortality in children and young adults. People over 60 years of age showed a higher prevalence of cross-reactive pH1N1 antibodies suggesting that they were previously exposed to an influenza virus or vaccine that was antigenically related to the pH1N1 virus. To define the basis for this cross-reactivity, we infected ferrets with H1N1 viruses of variable antigenic distance that circulated during different decades from the 1930s (Alaska/35), 1940s (Fort Monmouth/47), 1950s (Fort Warren/50), and 1990s (New Caledonia/99) and challenged them with 2009 pH1N1 virus six weeks later. Ferrets primed with the homologous CA/09 or New Jersey/76 (NJ/76) virus served as a positive control, while the negative control was an influenza B virus that should not cross-protect against influenza A virus infection. Significant protection against challenge virus replication in the respiratory tract was observed in ferrets primed with AK/35, FM/47, and NJ/76; FW/50-primed ferrets showed reduced protection, and NC/99-primed ferrets were not protected. The HAs of AK/35, FM/47, and FW/50 differ in the presence of glycosylation sites. We found that the loss of protective efficacy observed with FW/50 was associated with the presence of a specific glycosylation site. Our results suggest that changes in the HA occurred between 1947 and 1950, such that prior infection could no longer protect against 2009 pH1N1 infection. This provides a mechanistic understanding of the nature of serological cross-protection observed in people over 60 years of age during the 2009 H1N1 pandemic. Scientists from Emory University isolated a human monoclonal antibody (hMAb, EM4C04), highly specific for the 2009 pH1N1 virus hemagglutinin (HA), from a severely ill 2009 pH1N1 virus-infected patient. We postulated that under immune pressure with EM4C04, the 2009 pH1N1 virus would undergo antigenic drift and mutate at sites that would identify the antibody binding site. To test this hypothesis, we infected MDCK cells in the presence of EM4C04 and generated 11 escape mutants, displaying 7 distinct amino acid substitutions in the HA. Six substitutions greatly reduced mAb binding (K123N, D131E, K133T, G134S, K157N, G158E). Residues 131, 133 and 134 are contiguous with 157 and 158 in the globular domain structure contribute to a novel pH1N1 antibody epitope. One mutation near the receptor binding site, S186P, increased binding affinity of the HA to the receptor. 186P and 131E are present in the highly virulent 1918 HA and were recently identified as virulence determinants in a mouse-passaged pH1N1 virus. We found that pH1N1 escape variants expressing these substitutions enhanced replication and lethality in mice compared to wild-type 2009 pH1N1 virus. Increased virulence of these viruses was associated with an increased affinity for &#945;2,3 sialic acid receptors. Our study demonstrated that antibody pressure by a hMAb targeting a novel epitope in the Sa region of 2009 pH1N1 HA could inadvertently drive the development of a more virulent virus with altered receptor binding properties. These findings broaden our understanding of antigenic drift. There are several potential strategies for the development of vaccines to protect humans against influenza viruses, including formalin inactivated whole or split virus, HA subunit, and live attenuated virus vaccines. Live attenuated influenza vaccines (LAIV) have several attributes related to safety, immunogenicity, cross-protection against antigenic drift strains, high yield and needle-free administration that make them attractive candidates for control of pandemic influenza. Live attenuated vaccines generally induce broadly cross-reactive protection, which may be a useful feature in the event of a pandemic if a vaccine generated from the actual pandemic strain is not available. LID scientists collaborated with scientists from MedImmune under a CRADA to evaluate candidate vaccines against pandemic influenza viruses, including the 2009 pandemic H1N1 virus. The vaccine virus generated by MedImmune contains the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 H1N1 pandemic influenza virus and the attenuating genes from the A/Ann Arbor/6/60 cold adapted (A/AA/6/60 ca) donor virus. We had previously evaluated the efficacy and immunogenicity of a live attenuated cold adapted (ca) reassortant vaccine, A/California/7/2009 (CA09ca), in mice and ferrets. Although small animal models such as mice and ferrets are often used to study the pathogenesis of influenza viruses, these animals do not fully mirror the response to live attenuated influenza vaccines in humans. Therefore, we designed a study to evaluate the immunogenicity and protective efficacy of the live attenuated CA09ca pH1N1 vaccine in non-human primates. We found that replication of the CA09wt virus was different in two species of non-human primates; rhesus macaques supported replication of CA09wt and CA09ca viruses better than African green monkeys. In both species, the CA09wt virus replicated in the upper and lower respiratory tract, whereas replication of the CA09ca vaccine strain was severely restricted in the lower respiratory tract. We studied the immunogenicity and protective efficacy of the CA09ca virus in rhesus macaques, and found that vaccination with either 1 or 2 doses of vaccine elicited a protective antibody titer and conferred protection against challenge with the CA09wt virus.